Burkholderia pseudomallei, the causative agent of melioidosis and a potential bioterrorism threat, is intrinsically resistant to most commonly-prescribed antibiotics, demonstrating a clear requirement for alternative antimicrobials, as well as a better understanding of the pathogenic strategies utilized by this emerging organism. In Escherichia coli, an inter-bacterial contact-dependent inhibition (CDI) system comprised of two-partner secreted proteins was identified that inhibits target bacteria in an allele-specific manner. Toxicity, which is due to nuclease activity located at the C-terminal end of the CdiA exoprotein, is blocked by direct binding of the system's cognate immunity protein, CdiI. Homologs of this system are found in the genomes of B. pseudomallei and the related non-pathogen B. thailandensis. However, distinct differences exist between the Burkholderia and E. coli CDI systems and the role of these systems in Burkholderia biology is not known. Moreover, while a model for CDI has been suggested, the localization and topology of the CdiA/BtpA proteins are unknown, preventing a complete understanding of the CDI mechanism. Preliminary data indicate that the C-terminal region of the B. pseudomallei CdiA homolog, BtpA, which shares sequence similarity with colicin E5 tRNase, is toxic when produced in several bacterial species unless the system's cognate immunity protein, BtpI, is also produced. Toward the long-term objective of understanding the role of these systems in the lifecycle of B. pseudomallei, this application will test the hypothesis that BtpA is a surface-localized protein that functions as an intra- and/or inter-species nuclease to mediate inter-bacterial inhibition. Studies in Aim 1 will determine the topology and processing of the BtpA protein and experiments in Aim 2 will identify the mechanism of BtpA toxicity and define the minimal inhibitory unit. Finally, the research proposed in Aim 3 will assess the toxicity of BtpA to other bacterial pathogens and examine its ability to mediate intra- and/or inter- species bacterial inhibition. The proposed research will contribute to the understanding of an emerging pathogen and, by investigating a novel class of cytotoxic proteins, fulfills an essential need in the pursuit of novel antimicrobial therapies. PUBLIC HEALTH RELEVANCE: The bacterium Burkholderia pseudomallei is a global health problem and potential bioterrorism threat. By examining the function of antibacterial proteins produced by these bacteria, this research may lead to the development of new antimicrobial therapies against B. pseudomallei. More broadly, understanding how these and other pathogenic bacteria interact with each other will contribute to our ability to prevent transmission of infectious diseases.